The invention pertains to the field of regulating apoptosis in cells. In particular, the invention relates to the field of regulating apoptosis in cancerous and in non-cancerous cells.
Apoptosis, also referred to as xe2x80x9cprogrammed cell deathxe2x80x9d (PCD) is a natural phenomenon in which a series of events occurs that leads to the death of the cell. PCD is essential in the development of organisms and bodily tissues. PCD is also important in the removal from the body of potentially deleterious cells, such as cancer cells, virally infected cells, and autoimmune effector cells.
Cancer cells are often treated with agents that induce apoptosis, such as radiation or antineoplastic chemicals. Unfortunately, these apoptosis-inducing agents also exert their effects to some degree on non-neoplastic cells. This may result in the death of normal tissue adjacent to a cancer that is treated by radiation, or in serious local or systemic side effects when treating cancers with antineoplastic drugs.
An ideal anti-cancer therapeutic agent is one which kills cancer cells but which spares normal, non-cancerous cells. Such a therapy would permit the aggressive treatment of cancers without the risk of serious side effects. Consequently, a significant need exists for a method that protects normal, non-cancerous cells from undergoing apoptosis when exposed to apoptosis-inducing agents but which does not protect cancer cells, or at least which protects cancer cells to a lesser degree.
The inventors have discovered that the depletion of polyamines within a cell inhibits apoptosis in non-cancerous cells, but has a much decreased effect, if any, on apoptosis in cancerous cells. This discovery has important implications for anti-cancer therapy, such as chemotherapy, immunotherapy, and radiation therapy, in which agents that induce apoptosis are delivered to cells within the body. Typically, non-cancerous cells, as well as the target cancerous cells, are exposed to the apoptosis-inducing agents. By inhibiting apoptosis within non-cancerous cells while not inhibiting, or inhibiting to a lesser degree, apoptosis in cancerous cells, normal tissue may be spared during such treatment to kill cancer cells.
The invention is applicable for in vitro and in vivo experimental and therapeutic purposes. Cells, both normal and cancerous, may be from any animal, such as humans and domestic animals like dogs and cats. Likewise, patients that may benefit from the method of the invention may be human or veterinary patients, such as dogs and cats.
In one embodiment, the invention is a method for inhibiting apoptosis in normal, that is non-cancerous, cells which method includes the steps of depleting the level of polyamines within the cell and inhibiting apoptosis in the cell. In a preferred embodiment, the depletion of the level of polyamines within the cell is by inhibiting the intracellular enzyme ornithine decarboxylase (ODC). In a particularly preferred embodiment, ODC is inhibited by administering DL-œ-difluoromethylornithine (DFMO) to the cell. Preferably, the level of each of the polyamines; putrescine (PUT), spermidine (SPD), and spermine (SPM), are depleted, although in some situations in accordance with the method of the invention, PUT and SPD levels may be depleted and SPM levels may not be depleted.
In this specification, the term xe2x80x9cpolyaminexe2x80x9d refers to spermidine, spermine, and their diamine precursor, putrescine. For purposes of this specification the terms xe2x80x9cdepletedxe2x80x9d, xe2x80x9cdepletionxe2x80x9d, or xe2x80x9cdepletingxe2x80x9d, when referring to intracellular levels of any or all of the polyamines, PUT, SPD, and SPM, means that the level of the measurable polyamine within the non-cancerous cell is reduced sufficiently to inhibit apoptosis. Generally, intracellular PUT levels are reduced before SPD levels, which are reduced before SPM levels. In cell culture, PUT levels tend to become depleted within an hour, SPD within 2 days, and SPM after about 4 days following the initiation of treatment with an ODC inhibitor. The effect of depletion of polyamines in accordance with the invention to inhibit apoptosis in normal cells is usually observable within the first two days, even before the levels of SPM begin to drop. Similar patterns are observed with in vivo ODC inhibition therapy in live animals.
In accordance with the method of the invention, measurable PUT in the cell is generally reduced by 50% or more, preferably by 80% or more, and most preferably by 100% from the pre-treatment level. SPD levels are typically reduced by 30% or more, preferably by 50% or more, and most preferably by 80% or more from the pre-treatment level. SPM levels may not be reduced by the time that an inhibition of apoptosis in accordance with the method of the invention is observed. Generally, beginning several days, such as 2 to 4 days, following the initiation of therapy, SPM levels are reduced by 10% or more, preferably by 30% or more, and most preferably by 50% or more from the pretreatment level. However, the actual level of reduction of each or all of the polyamines is immaterial, so long as the level of polyamines is reduced sufficiently to inhibit apoptosis in normal cells, that is to achieve depletion of the polyamines.
In another embodiment, the invention is a method for selectively inhibiting apoptosis in normal, non-cancerous cells, while not inhibiting apoptosis, or inhibiting apoptosis to a lesser degree, in cancerous cells. The method of the invention according to this embodiment includes administering to a patient suffering from a cancer an effective amount of an agent that causes the depletion of the level of polyamines within the normal, non-cancerous cells, and inhibiting apoptosis in the normal cells, and which agent inhibits apoptosis to a lesser extent or not at all in the cancerous cells. In a preferred embodiment, the depletion of the level of polyamines is by inhibiting ODC. In a particularly preferred embodiment, ODC is inhibited by administering DFMO to the patient. Preferably, the level of each of the polyamines; putrescine (PUT), spermidine (SPD), and spermine (SPM), are depleted, although as stated above, levels of intracellular PUT are depleted before the levels of SPD, which are in turn depleted before the levels of SPM, and apoptosis in normal tissues may be inhibited before depletion of all the polyamines.
In accordance with the invention, the depletion of the polyamines typically occurs in cancerous cells, similarly to the depletion seen in normal cells. However, unlike what occurs with normal cells, the depletion of polyamines in cancerous cells does not result in an inhibition of apoptosis.
In another embodiment, the method of the invention is a method for treatment of a patient with cancer, which method includes administering to the patient an effective amount of an agent that causes the depletion of polyamines within cells, such as an inhibitor of ODC, depleting the polyamines in the patient""s cells, and exposing cancer cells and normal cells within the body of the patient to an apoptosis-causing agent, thereby killing the cancer cells and not killing the normal cells, or killing the normal cells at a reduced rate compared to the cancer cells. The patient may be any animal, such as a human or a veterinary patient, including dogs, cats, and ferrets, and livestock such as cattle, horses, goats, sheep, and pigs. The apoptosis-causing agent may be anything that induces apoptosis, such as a chemical agent, like an antineoplastic drug, an immunotherapeutic agent, or an electromagnetic or acoustic ray, such as an x-ray or gamma ray. Preferably, the ODC inhibitor is DFMO.